Detection of somatic variants from next-generation sequencing data in grapevine bud sports

The grapevine (Vitis vinifera) is one of the oldest and the most valuable horticultural crops. Sexual crossing has been a major driver of grapevine evolution and, more recently, it has generated thousands of varieties. Somatic variation plays a crucial role in intravarietal grapevine diversity, generating novel interesting phenotypes. Somatic mutations that accidentally happened in buds of vegetatively propagated varieties were frequently noticed and the resulting bud sports were selected for their distinguished phenotype. In this work, we aimed to explore clonal variability to identify DNA mutations and transcriptional changes among genomes within a grapevine variety. Vitis vinifera is an ideal model because there are many clones with visible phenotypic differences and a high quality reference sequence is available (Jaillon et al 2007). Previous studies of clonal diversity used SSR and AFLP markers that only enabled the identification of a limited number of clones. Thus, we adopted a whole genome scan approach. Illumina next generation sequencing technology was used to resequence four ‘Pinot’ clones (‘Pinot blanc’, ‘Pinot gris’, ’Pinot Meunier’ and ‘Pinot noir’) and two ‘Sangiovese’ clones (commercially called ‘R24’ and ‘VCR23’). Post-processed paired-end reads (2x100bp) were mapped against the PN40024 reference genome obtaining a depth of coverage >35x. Four libraries were of high quality, while the distribution of 16-kmers occurrences in the ‘Pinot gris’ and ‘Pinot noir’ Illumina reads revealed low complexity of the library and suggested to discard those clones for subsequent analyses. SNPs were first detected in the pairwise comparison ‘Pinot blanc’ and ‘Pinot Meunier’ using the GATK – UnifiedGenotyper tool with default parameters, followed by a quality filtering and by a calibration step. In the filtering step, we removed SNPs in repetitive regions, transposable elements, and regions surrounding microsatellite motifs and INDELs, we removed bad quality SNPs based on GATK internal parameters, SNPs with 3-fold the average coverage). The calibration step was based on quality scores of a known somatic variation in ‘Pinot Meunier’ in the position chr1:4,897,066 (Boss and Thomas 2002). In the comparison between ‘Pinot blanc’ and ‘Pinot Meunier’ we ended up with a total of 144 putative SNPs, 79 of which were validated as true positive by Sanger resequencing (29 in ‘Pinot blanc’ and 50 in ‘Pinot Meunier’) with a FDR of 0.33 and 0.24, respectively. We performed the pairwise comparison between ‘Sangiovese R24’ and ‘Sangiovese VCR23’ with the same parameters used for ‘Pinot’ clones, ending up with only three putative variant positions. Of these, two SNPs were validated as true positive by Sanger resequencing. In all cases, Sanger resequencing confirmed the chimerical nature of the putative somatic mutation. Genome scanning for copy number variations larger than 25 kbp was performed by a depth of coverage (DOC) analysis and revealed only the known somatic deletion in ‘Pinot blanc’ in the interval chr2:14,149,000..14,250,000 as compared to ‘Pinot Meunier’. The complementary approach of paired-end mapping (PEM) revealed 11 putative deletions smaller than 25kbp in ‘Pinot blanc’, 19 in ‘Pinot gris’, 15 in ‘Pinot Meunier’, and 5 in ‘Pinot noir’ as unique to each clone and not shared with a set of 20 varieties of Vitis vinifera analysed with the same pipeline. In the comparison of ‘Sangiovese’ clones, the PEM algorithm identified seven putative deletions in ‘Sangiovese VCR23’, not shared with either ‘Sangiovese R24’ or other varieties of Vitis vinifera. No copy number variation larger than 25 kbp was detected by a depth of coverage (DOC) analysis between ‘Sangiovese’ clones. We also compared the transcriptome of different clones in order to monitor gene expression changes that could be directly or indirectly related to somatic mutations at the DNA level. We obtained RNA-seq of leaf tissues of the same ‘Pinot’ and ‘Sangiovese’ clones analysed by DNA sequencing. Furthermore for ‘Sangiovese’ clones, we sequenced berry transcriptomes at two developmental stages – before ripening (2 weeks after berry set) and at the inception of ripening. More than 30,000 genes were expressed in all clones of both varieties. The vast majority of the predicted genes in the grapevine genome was transcribed at detectable levels in all organs and stages of development investigated. Under the same experimental conditions, leaf transcriptomes were much more variable in pairwise comparisons between ‘Pinot’ clones than between the pair of ‘Sangiovese’ clones. Between the clones of ‘Sangiovese’, the widest differentiation in terms of global transcriptome was detected in berries collected two weeks after fruit set. Genes that showed significant differences in transcriptional levels between clones were in general not correlated with the position of the DNA mutations identified by DNA sequencing. Through the power of the Next Generation Sequencing technology we have produced a sufficient depth and breadth of sequence coverage to comprehensively discover somatic mutations that allowed us to distinguish four ‘Pinot’ clones and two ‘Sangiovese’ clones analysed in this study. At the DNA level, somatic mutations in two ‘Sangiovese’ genomes appeared to be more rare than those observed among ‘Pinot’ clones, which corresponds to a lower level of phenotypic differentiation between the two ‘Sangiovese’ clones and is in accord with a presumed more recent origin compared to the ‘Pinot’ clones. This analysis provides the first whole-genome estimation of the rate of somatic mutation in grapevine varieties.Struttura di aggregazione: Istituto di Genomica Applicata - Co-supervisor: Gabriele Di GasperoopenDottorato di ricerca in Scienze e biotecnologie agrarieembargoed_20141009Miculan, Mar

Genome wide association study of agronomic and seed traits in a world collection of proso millet (Panicum miliaceum L.)

The climate crisis threatens sustainability of crop production worldwide. Crop diversification may enhance food security while reducing the negative impacts of climate change. Proso millet (Panicum milaceum L.) is a minor cereal crop which holds potential for diversification and adaptation to different environmental conditions. In this study, we assembled a world collection of proso millet consisting of 88 varieties and landraces to investigate its genomic and phenotypic diversity for seed traits, and to identify marker-trait associations (MTA)

Transcriptome and metabolite profiling reveals that prolonged drought modulates the phenylpropanoid and terpenoid pathway in white grapes (Vitis vinifera L.)

Background: Secondary metabolism contributes to the adaptation of a plant to its environment. In wine grapes, fruit secondary metabolism largely determines wine quality. Climate change is predicted to exacerbate drought events in several viticultural areas, potentially affecting the wine quality. In red grapes, water deficit modulates flavonoid accumulation, leading to major quantitative and compositional changes in the profile of the anthocyanin pigments; in white grapes, the effect of water deficit on secondary metabolism is still largely unknown Results: In this study we investigated the impact of water deficit on the secondary metabolism of white grapes using a large scale metabolite and transcript profiling approach in a season characterized by prolonged drought. Irrigated grapevines were compared to non-irrigated grapevines that suffered from water deficit from early stages of berry development to harvest. A large effect of water deficit on fruit secondary metabolism was observed. Increased concentrations of phenylpropanoids, monoterpenes, and tocopherols were detected, while carotenoid and flavonoid accumulations were differentially modulated by water deficit according to the berry developmental stage. The RNA- sequencing analysis carried out on berries collected at three developmental stages—before, at the onset, and at late ripening—indicated that water deficit affected the expression of 4,889 genes. The Gene Ontology category secondary metabolic process was overrepresented within up-regulated genes at all the stages of fruit development considered, and within down-regulated genes before ripening. Eighteen phenylpropanoid, 16 flavonoid, 9 carotenoid, and 16 terpenoid structural genes were modulated by water deficit, indicating the transcriptional regulation of these metabolic pathways in fruit exposed to water deficit. An integrated network and promoter analyses identified a transcriptional regulatory module that encompasses terpenoid genes, transcription factors, and enriched drought- responsive elements in the promoter regions of those genes as part of the grapes response to drought Conclusion: Our study reveals that grapevine berries respond to drought by modulating several secondary metabolic pathways, and particularly, by stimulating the production of phenylpropanoids, the carotenoid zeaxanthin, and of volatile organic compounds such as monoterpenes, with potential effects on grape and wine antioxidant potential, composition, and sensory feature

Data-driven, participatory characterization of farmer varieties discloses teff breeding potential under current and future climates

In smallholder farming systems, traditional farmer varieties of neglected and underutilized species (NUS) support the livelihoods of millions of growers and consumers. NUS combine cultural and agronomic value with local adaptation, and transdisciplinary methods are needed to fully evaluate their breeding potential. Here, we assembled and characterized the genetic diversity of a representative collection of 366 Ethiopian teff (Eragrostis tef) farmer varieties and breeding materials, describing their phylogenetic relations and local adaptation on the Ethiopian landscape. We phenotyped the collection for its agronomic performance, involving local teff farmers in a participatory variety evaluation. Our analyses revealed environmental patterns of teff genetic diversity and allowed us to identify 10 genetic clusters associated with climate variation and with uneven spatial distribution. A genome-wide association study was used to identify loci and candidate genes related to phenology, yield, local adaptation, and farmers' appreciation. The estimated teff genomic offset under climate change scenarios highlighted an area around lake Tana where teff cropping may be most vulnerable to climate change. Our results show that transdisciplinary approaches may efficiently propel untapped NUS farmer varieties into modern breeding to foster more resilient and sustainable cropping systems

The genomic and bioclimatic characterization of Ethiopian barley (Hordeum vulgare L.) unveils challenges and opportunities to adapt to a changing climate

The climate crisis is impacting agroecosystems and threatening food security of millions of smallholder farmers. Understanding the potential for current and future climatic adaptation of local crop agrobiodiversity may guide breeding efforts and support resilience of agriculture. Here, we combine a genomic and climatic characterization of a large collection of traditional barley varieties from Ethiopia, a staple for local smallholder farmers cropping in challenging environments. We find that the genomic diversity of barley landraces can be partially traced back to geographic and environmental diversity of the landscape. We employ a machine learning approach to model Ethiopian barley adaptation to current climate and to identify areas where its existing diversity may not be well adapted in future climate scenarios. We use this information to identify optimal trajectories of assisted migration compensating to detrimental effects of climate change, finding that Ethiopian barley diversity bears opportunities for adaptation to the climate crisis. We then characterize phenology traits in the collection in two common garden experiments in Ethiopia, using genome-wide association approaches to identify genomic loci associated with timing of flowering and maturity of the spike. We combine this information with genotype–environment associations finding that loci involved in flowering time may also explain environmental adaptation. Our data show that integrated genomic, climatic, and phenotypic characterizations of agrobiodiversity may provide breeding with actionable information to improve local adaptation in smallholder farming systems

Matthew 10,16 - exegesis and history of interpretation

Matthew 10.16, which uses metaphorical statements known from Greek and Jewish literature, stands at the center of the second great discourse of the Gospel of Matthew (missionary speech) as a bridge between the first and second parts, which briefly summarizes its basic themes − Jesus' sending the disciples, their situation in a hostile world and features needed to fulfill this task in these conditions. In my own translation, I decided to keep the words (omitted in some translations) that have an important function in the text − "behold" (as macrosyntactical signal, which focuses attention on new important theme), "I" (as emphasis on the person of a speaker who is at once a Shepherd, Sheep and Lamb) and "therefore" (as emphasis on necessity of these properties which follows from the facts mentioned in the previous sentence − both from their task and their position as disciples in a hostile world). I prefer the word "pure" for the translation of properties of doves, which (in my opinion) aptly expresses the importance of both textual variants without the negative connotations of the word "simple" (as naive or stupid). Prudence is not only a way to avoid danger, but rather finding ways to fulfill the task. Purity means that the disciple is not adapt to this world (to be a wolf among wolves), but follow..

Additional file 9: Figure S5. of Transcriptome and metabolite profiling reveals that prolonged drought modulates the phenylpropanoid and terpenoid pathway in white grapes (Vitis vinifera L.)

Carotenoid network. Network representation of carotenoids in C (A) and D (B) berries during development. Nodes represent ‘metabolites’ and edges represent ‘relationships’ between any two metabolites. Edges colored in red and blue represent significant (P < 0.001) positive and negative correlations, respectively. Metabolites in bold indicate a significant effect (P < 0.05) of water deficit on the concentration of that metabolite at one or more developmental stages. Number of correlating edges, average node neighborhood, and clustering coefficient were similar between C and D networks. (PNG 63 kb